Fluorometirc enzyme inhibition immunoassay for measuring potency of allergen extracts

ABSTRACT

An inhibition assay for measuring the potency of allergen extracts by incubating a mixture of allergen extract and reference allergen specific IgE in a buffered solution with an insoluble support to which reference allergen is adhered. The conjugated IgE adhering to the insoluble support is reacted with an enzyme labeled anti-IgE antibody and the enzyme label is contacted with a solution of a substrate which will yield a fluorescent product in the presence of the enzyme. The level of fluoresence in the solution is measured. The percentage of inhibition of the allergen specific IgE is determined from fluorescence levels measured for various extract concentrations.

RELATIONSHIP TO COPENDING APPLICATIONS

This application is a Continuation-in-Part of applications Ser. No.444,622 filed Nov. 26, 1982 and Ser. No. 434,061 filed Oct. 13, 1982.

FIELD OF THE INVENTION

This invention relates to methods for measuring potency of allergenicextracts with increased precision. In particular, this invention relatesto procedures for calibrating the potency of allergenic extracts againstpotency standards so that the extracts can be used with greaterprecision and reliability in desensitization treatments of patients forallergic reactions.

BACKGROUND OF THE INVENTION DESCRIPTION OF THE PRIOR ART

Radiometric and fluorometric methods for identifying and measuringallergy specific IgE levels in patient serum are commercially availableand are known as the RAST test, for example. U.S. Pat. Nos. RE-29,474;3,555,143; 3,648,346; 3,720,760 and 3,966,898 relate to these methodsand reagents therefor. Enzymatic immunological methods for identifyingand quantifying antigens and antibodies in liquids are widely used andare known as the ELISA and EIA tests, for example. Basic technology forenzymatic assays and reagents therefor are disclosed in U.S. Pat. Nos.RE-29,169 and 3,839,153, for example.

A review of the current state of the art with regard to immunoassays forthe detection of proteins in solutions is provided by R. Rose et al,Manual of Clinical Immunology, 2nd ed. American Society forMicrobiology, Washington, pp 327-429, 775-849 (1980) and by A. Voller etal, Immunoassays for the 80's, University Park Press, Baltimore (1981),and the publications cited in both of these publications, the entirecontents all of which are hereby incorporated by reference. The chapterof Immunoassays for the 80's, supra, by T. A. E. Platts-Mills et al,titled "Radio-immunoassays in Allergy", pp 289-311, and the publicationscited therein provide a comprehensive review of allergy tests.

Procedures for standardizing allergens are summarized by Rose et al inManual of Clinical Immunology, supra, pp 778-788, and the citationstherein. In one RAST approach, allergen of unknown potency is coupled toa solid support, and the quantity required to give a certain degree ofreactivity in the RAST test has been determined. In a second approachtermed "RAST inhibition", the allergen of unknown potency is incubatedwith standardized allergen coupled to a solid support and reagent IgEspecific to the allergen, and competitive binding occurs. In bothprocedures, the bound IgE is determined with radiolabeled IgG specificto IgE, and the results are compared to those obtained with theprocedure using reference allergens of known potency. Both procedureshave limited accuracy, however. Partially because of the widediscrepancies found in tests of the same material (test limitations) andthe crude, highly variable extracts of very limited stability usuallyadministered (reagent variability), mandatory potency standards have notyet been adopted by regulatory agencies.

Procedures for binding proteins to insoluble supports have beenpreviously described. Antibodies have been covalently bonded toinsoluble supports as described in U.S. Pat. Nos. 3,551,555, 3,553,310,4,048,298 and RE-29,474. Binding of antibodies to polystyrene byadsorption has been described in U.S. Pat. Nos. 3,646,346 and 4,092,408,for example. Allergens have been covalently bonded to a variety ofinsoluble supports as described in U.S. Pat. No. 3,720,760.

Polyethylene glycol has been used in protein fractionation processesdescribed by A. Polson et al, Biochim. Biophys Acta, vol. 82, pp 463-475(1964) and A. Polson et al, Vox Sang, vol. 23, pp. 107-118 (1972).

SUMMARY OF THE INVENTION

This invention relates to a method for measuring the potency of allergenextracts. It comprises the steps of first contacting an insolublesupport having reagent allergen adhered thereto with an aqueous solutioncontaining a known concentration or dilution of the allergen extract anda known amount of IgE specific to allergenic components present in thereagent allergen and presumed present in the allergen extract. Theamount of IgE is insufficient to bind with all of the reagent allergen.The contact is maintained for a time sufficient to permit conjugation ofIgE with allergenic components. The solution is then removed from thesupport. The support is then contacted with an aqueous solution ofanti-IgE antibody labeled with a fluorogenic enzyme, for sufficient timeto permit conjugation of anti-IgE antibody with IgE which has conjugatedwith reagent allergen on the insoluble support. The term "fluorogenicenzyme", as used herein means an enzyme, in the presence of which, asubstrate will release or react to yield a fluorescent product. Thesolution is then removed from the support. The support is then contactedwith an aqueous solution of substrate which undergoes chemical reactionto yield a fluorescent product when in the presence of the fluorogenicenzyme, for sufficient time to yield a fluorescent product. The level offluorescence in the solution is then measured.

In certain preferred embodiments of this invention, the insolublesupport has a plurality of test wells separated by opaque material, theanti-IgE antibody is a monoclonal antibody to which alkaline phosphataseis bound, the anti-IgE is contacted with the insoluble support in anaqueous solution containing from 1 to 8 weight percent polyethyleneglycol having a molecular weight of from 1000 to 10,000 and a non-ionicsurfactant, and the substrate is 4-methylumbelliferyl phosphate. If theallergen bonded to the insoluble support is covalently bonded to awater-soluble protein having absorption or adsorption affinity for theinsoluble support, the insoluble support is preferably prerinsed with anaqueous buffered rinse solution containing from 0.0001 to 0.5 weightpercent of the water-soluble protein.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE is a graph showing inhibition curves for allergens ofreference and unknown potencies.

DETAILED DESCRIPTION OF THE INVENTION

Key to successful treatment of allergic conditions is the accurateidentification of the offending allergen and the titration of thepatient to determine the effective desensitization dosage. In general,reconstituted allergen extract is injected in sufficient quantity tocause major production of antigen-specific IgG (blocking antibody) andmajor production and/or activation of suppressor T lymphocytes. However,the quantity should not be sufficient to cause major allergic reaction.To the extent that antigen-specific IgE is produced at an increasedlevel, it is critical that the IgG and suppressor IgE production be insuch balance as to prevent allergic reaction.

The concentration and amount of the desensitization dosage are dependentupon many factors which are specific to the patient undergoing theallergic reaction. It is, therefore necessary to titrate the patient todetermine the proper dosage. A variety of standard titrating techniquesare available. Examples of traditional procedures are described inRemington's Pharmaceutical Sciences, supra, pp 1344-1352, the entirecontents of which are incorporated herein by reference. However, themethods available prior to this invention have lacked the specificityand accuracy to be more than a rough approximation of the order ofmagnitude of the appropriate beginning dosage range.

Methods providing the specificity and accuracy to determine a suitabledesensitization dosage, particularly when the allergen used fordesensitization and the allergen component of the diagnostic method havethe same allergen profile and specificity have been described in ourcommonly assigned, copending application Ser. No. 462,585 filed Jan. 31,1983, the entire contents of which are incorporated by reference. Afteridentification and quantification of the offending allergen, standarddesensitization immunotherapy procedures are employed. The procedurenormally used involves injecting into the patient gradually increaseddoses of the allergen, usually to maximum tolerated doses (doses notgiving rise to major allergic response), at varying intervals in anattempt to develop IgG antibody protection against the agents and toincrease the specific suppressor T lymphocyte activity.

With the method of this invention, the potency of the allergenic extractcan be precisely measured, permitting an exact determination of thedosage of extract which should be administered, making unnecessary theexacting procedures formerly required. The exact mechanisms of thistreatment are not fully understood. Booster injections to maintain therequisite IgG and suppressor T lymphocyte levels are required atintervals of one to four weeks. Usually the doses required for boosterinjections are substantially greater than the maximum dose required forcontrol of the initial allergic reaction.

Comparable activity levels of two allergen extracts can be determined bycomparing allergen activity levels of serial dilutions of one allergenextract with serial dilutions of the other allergen extract. Thecomparative dilutions at which one allergen extract has the same potencyas the other extract indicate the comparative potencies. This procedurecan be carried out using the novel fluorometric inhibition assay methodof this invention. If one of the extracts is an allergen extract to beused as a standard for assigning a relative potency, the relativepotency expressed as relative dilution required to match the potency ofthe standard can be determined. Because of the improved accuracy andreproducability provided by the assay of this invention and theavailability of highly purified, stable allergens which is the subjectof concurrently filed, commonly assigned patent application Ser. No.476187 AX-19 filed 3-17-83 by Tsay Yuh-geng, Myron A. Beigler, EmanuelCalenoff, Gerald L. Friesen and James L. Nichols, entitled StableAllergenic Extracts And Methods, it is possible to provide allergens ofstandardized potencies for diagnostic and therapeutic purposes.

The fluorometric inhibition assay method of this invention comprises afirst step of contacting an insoluble support having reagent allergenadhering thereto with an aqueous solution. The solution contains a knownconcentration or dilution of allergen extract and a known amount ofreagent IgE specific to the allergenic components present in the reagentallergen and presumed present in the allergen extract. The amount ofreagent IgE is insufficient to bind with all of the reagent allergen.The contact is maintained for a time sufficient to permit conjugation ofreagent IgE with allergenic components. The solution is then removedfrom the support.

The contact or incubation time should be sufficient to permitsubstantial conjugation to occur between reagent Ige and allergen, thetime being temperature dependent. Suitable incubation times are from 30to 180 minutes at temperatures within the range of from 18° to 40° C.,the preferred contact time being from 60 to 120 minutes at temperatureswithin the range of from 20° to 26° C.

The solution contains an amount of reagent Ige specific to theallergenic components which is insufficient to bind with all of theallergenic components present in the reagent allergen adhering to theinsoluble support. Preferably the amount is sufficient to bind from 5 to20 and optimally from 8 to 10 times the binding capacity of an IgE freecontrol serum. Allergen specific reagent IgE is known in the art and ismost conveniently obtained as a serum pool from atopic patients withallergen specific IgE.

The solution preferably contains buffers. The preferred solution has apH of from 6 to 8 and can be a phosphate buffer solution having aphosphate molarity of from 0.01 to 0.05 and contains from 0.01 to 0.1weight percent non-ionic surfactant, and from 0.0001 to 0.5 of anon-interfering animal protein.

The insoluble support having the allergen adhering thereto is animportant aspect of this invention. The allergen can be any allergenicmaterial such as allergen derived from pollens derived from trees,shrubs, weeds, and grasses; molds; smuts; dusts; allergens derived fromdanders, hair, and epidermals of animals; extracts derived from insectsincluding insect venoms; and from foods.

A wide variety of compounds can be employed as the solid support, theprimary consideration being the binding of the allergens to the surface,the absence of interference with the enzyme labeled anti-IgE antibodyreagent, enzymatic reaction thereof with a substrate and fluorescentproperties of the enzymatic reaction product. Suitable supports and thecoupling of allergens thereto are described in commonly assigned,copending application Ser. No. 462,585 filed Jan. 31, 1983.

A preferred diagnostic support of this invention comprises apolystyrene, styrene copolymers including styrene-(vinyl monomer)copolymers such as styrene-acrylonitrile copolymers, or polyolefins suchas polyethylene and polypropylene, and acrylate and methacrylatepolymers and copolymers in the form of beads or other surfaces. Theallergenic extract is preferably bound thereto by adsorption, ionicbonding, van der Waals adsorption, electrostatic bonding, othernon-covalent bonding. It can also be bound to the support by covalentbonding. A particularly advantageous support for this procedurecomprises a microtiter plate having a plurality of wells. The wellsurface or plastic cup inserts therein can constitute the allergensupport. Most advantageously, the microtiter plate or the well insertsare opaque to light so that excitation light applied to a well orfluorescence generated in response thereto does not reach or influencecontents of the surrounding wells. With this system each well can beemployed as a test system independent of the other wells.

Preferably the allergen is covalently bonded to a water-soluble polymerhaving an affinity for the insoluble substrate. The allergen-polymerproduct is then adhered to the insoluble substrate by non-covalentbonding such as by adsorption or absorption.

Suitable water-soluble proteins include bovine serum albumins of bovine(BSA), human (HSA), rabbit (RSA), goat (GSA), sheep (SSA), horse (HOSA),etc.; serum gamma Globulin of the previously described animals; andother animal proteins such as ovalbumin, fribrinogen, thrombin,transferin, glycoproteins, etc. Suitable water-soluble amino acidpolymers include polylysine, polyglutamic acid, polyalanine,polyhistidine, polymethionine, polyproline, etc. The allergen can becovalently bonded to water-soluble protein or amino acid polymer withconventional coupling agents using methods which are known in the art.Preferably the coupling agent is a carbodiimide such as1-ethyl-3-(3-N,N-dimethylaminopropyl)carbodiimide hydrochloride and1-cyclohexyl-3(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate.Other suitable coupling agents include aldehyde coupling agents havingeither ethylenic unsaturation such as acrolein, methacrolin, or2-butenal or having a plurality of aldehyde groups such asglutaraldehyde, propanedial or butanedial. Other coupling agents includebifunctional NHS-esters such as disuccinimidyl suberate, disuccinimidyltartarate, bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone,disuccinimidyl(N,N'-diacetylhomocystine, dithiobis(succinimidylpropionate), ethylene glycolbis(succinimidyl succinate);heterobifunctional reagents such as N-5-azido-2-nitrobenzoyloxysuccinimide, p-azidophenacyl bromide, p-azidophenylglyoxal,4-fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoate,m-maleimidobenzoyl N-hydroxysuccinimide ester,methyl-4-azidobenzoimidate.HCl, p-nitrophenyl2-diazo-3,3,3-trifluoropropionate,N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate, succinimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate, succinimidyl4-(p-maleimidophenyl)butyrate,N-succinimidyl(4-azidophenyldithio)propionate, N-succinimidyl3-(2-pyridyldithio)propionate, N-(4-azidophenylthio)phthalimide,homobifunctional reagents such as 1,5-difluoro-2,4-dinitrobenzene,4,4'-difluoro-3,3'-dinitrodiphenylksulfone,4,4'-diisothiocyano-2,2'-disulfonic acid stilbene,p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenylester), 4,4'-dithiobisphenylazide, erythritolbiscarbonate; andbifunctional imidoesters such as dimethyl adipimidate.2HCl, dimethylsuberimidate, dimethyl 3,3'-dithiobispropionimidate.2HCl,2-iminothiolane.HCl. Covalent bonding of allergen to the insolubleprotein can be carried out with the above reagents by conventional,well-known reactions, for example in the aqueous solutions at a neutralpH, at temperatures of less than 10° C. for 18 hours or overnight.

In a procedure for non-covalent adhesion of allergen to the surface ofan insoluble support, the allergenic material can be applied to thesurface of a support such as a polystyrene microtiter well orpolystyrene individual insert well therefor, in an aqueous buffersolution. The surface is initially cleaned with a cleaning fluid such asmethanol and dried. The buffered allergen solution is placed in the wellor insert cup and incubated at room temperature until adsorption occurs,for example for from 2 to 18 hours and preferably from 16-18 hours, attemperatures of from 4° to 40° C. and preferably from 20° to 26° C. Thewell is then rinsed with a weak saline solution and dried.

Procedures for covalently adhering allergens to insoluble supports aredescribed by Ichiro Chibata in Immobilized Enzymes, Halsted Press, NewYork, 1978, and by A. Cuatrecasas, J. Bio. Chem. 245 3059(1970), theentire contents of which are hereby incorporated by reference. Thesurface can be coated with a protein and coupled with allergen using theprocedures described in U.S. Pat. No. 4,210,418 using glutaraldehyde asa coupling agent, for example. In a still further procedure, the wellcan be coated with a layer having free isocyanate groups such as apolyether isocyanate, and application of the allergen in aqueoussolution thereto effects the requisite bonding. In a still furtherprocedure, the allergen can be coupled to a hydroxylated material bymeans of cyanogen bromide as described in U.S. Pat. No. 3,720,760.

Preferred allergens are those described in commonly assigned copendingpatent application Ser. No. 433,962 filed Oct. 13, 1982, the entirecontents of which are incorporated by reference.

If the allergen is covalently bonded to a water-soluble polymer havingan affinity for the insoluble substrate and the water-soluble polymerhas antigenic properties, the first step is preferably preceded by aprerinse step. In the prerinse step, the support surface is contactedwith an aqueous buffered rinse solution containing from 0.0001 to 0.5weight percent of the water-soluble antigenic polymer to which theallergen is bound. This prerinse step is particularly advantageous whenthe water-soluble polymer is water-soluble animal protein because rinseresidue will provide a sufficient amount of the water-soluble protein toconjugate with any of the protein-specific IgE which might be present inthe solution.

A preferred rinse solution of this invention is an aqueous phosphatebuffer solution having a phosphate molarity of from 0.0001 to 0.05, a pHof from 6 to 8 and containing from 0.001 to 0.1 weight percent non-ionicsurfactant and from 0.0001 to 0.5 weight percent of the antigenicprotein to which the allergen is coupled. Suitable non-ionic surfactantsinclude polyoxyethylene eithers (BRIJ®) such as lauryl, cetyl, oleyl,stearyl, and tridecyl polyoxyethylene ethers; polyoxyethylenesorbitans(TWEEN®) such as polyoxyethylenesorbitan monolaurate, monopalmitate,monostearate, monoleate and trioleates; and other polyoxyethylene ethers(TRITON®), for example. A preferred non-ionic surfactant isoctylphenoxypolyethoxy ethanol having 40 ethylene oxide units (TRITONX-405, Rohm and Haas Company.

The buffer solution is advantageously prepared from a reagentconcentrate of the invention comprising from 0.005 to 2.5 weight percentof the animal protein corresponding to the animal protein to which theallergen is covalently bonded, from 0.5 to 5 weight percent non-ionicsurfactant, from 10 to 20 weight percent sodium chloride, from 0.5 to 5weight percent stabilizer and sufficient phosphate salt to provide for a0.02 to 0.05 M phosphate solution. The pH can be from 6 to 8. Thepreferred buffer concentrate contains about 0.5 weight percent of theanimal protein, 0.1 weight percent TRITON X-405 non-ionic surfactant, 17weight percent sodium chloride, and 2 weight percent sodium azide, 0.01Mphosphate and has a pH of 7.4.

After conjugation of reagent IgE with allergen adhering to the insolublesupport has occurred, the solution is removed therefrom. Surplus liquidis removed and the solid surface is then rinsed with a suitable rinsesolution such as that described above.

The second step of the process of this invention comprises contactingthe insoluble support with an anti-Ige antibody labeled with afluorogenic enzyme. The incubation is continued for sufficient time topermit serum IgE conjugated with allergen (if any) on the insolublesupport to conjugate with the anti-IgE antibody. After incubation, theexcess liquid is removed, and the surface of the insoluble support isrinsed with a weak saline solution as described above with respect tothe first step to remove unconjugated antibody. Preferably the supportis rinsed with the preferred rinse solution of this invention describedabove.

Anti-IgE antibodies are available from many sources, and the methodologyfor producing them is well known and is described in several of thepatents and publications cited above. The preferred antibodies aremonoclonal antibodies. The technology for making monoclonal antibodiesis well developed, and the procedures suitable for making monoclonalanti-IgE antibodies are described by D. Catty, et al in "Antisera inImmunoassays with special Reference to Monoclonal Antibodies to HumanImmunoglobulins", Immunoassay's for the 80's, supra, pp 133-153 and thepublications cited therein, the entire contents of which are herebyincorporated by reference.

Fluorogenic enzymes (enzymes in the presence of which a substrate willproduce a fluorescent product) and methods for bonding enzymes toantibodies without impairing the ability of the antibody to selectivelyconjugate with antigen are well known in the art. Suitable enzymes andprocedures for coupling them to antibodies are described in U.S. Pat.No. 4,190,496, for example, the contents thereof being herebyincorporated by reference. The preferred fluorogenic enzymes and thesuitable substrates corresponding thereto include horse-radishperoxidase for which a suitable substrate is homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, beta-galactosidase for which asuitable substrate is 4-methylumbelliferyl-beta-D-Galactoside, alkalinephosphatase for which a suitable substrate is 4-methylumbelliferylphosphate and other umbelliferyl phosphates such as 4-carboxyumbelliferyphosphate, and umbelliferyl phosphate 4-carboxy alkylesters, etc.

Examples of suitable procedures for enzyme labeling the anti-IgEantibody include the use of carbodiimides, dialdehydes, and bifunctionalcoupling reagents as described above for covalently bonding allergen towater-soluble proteins such as BSA, for example.

The enzyme labeled anti-IgE antibody is applied to the insoluble supportin an aqueous solution. The solution preferably contains suitable saltsand buffers to preserve the reactants and facilitate the conjugationreaction. For example, the solution can contain bovine serum albumin(BSA), phosphate buffer solution (PBS), and a mild surfactant such as apolyoxyethylene sorbitan ester employed in the rinse solutions describedabove. The rinse solutions described herein can also be used.

A preferred solution of this invention comprises from 0.1 micrograms perml to 5 micrograms per ml and preferably from 1 microgram per ml to 2microgram per ml anti-IgE antibody in an aqueous phosphate bufferedsolution having a phosphate molarity of from 0.005 to 0.1 and preferablyfrom 0.0001 to 0.05 and a pH of from 6.0 to 8.0 and preferably 7.2 to7.6. A critical ingredient in the anti-IgE solution is polyethyleneglycol having molecular weights of from 1000 to 8000 and preferably from2000 to 4000 in concentrations of from 1 to 8 weight percent andpreferably from 2 to 6 weight percent. Polyethylene glycols greatlyincrease the speed and sensitivity of the reaction. Another importantingredient is a non-ionic surfactant in concentrations of from 0.001 to0.5 and preferably from 0.02 to 0.1 weight percent. Suitable non-ionicsurfactants include those described above with respect to rinsesolutions, for example. A preferred non-ionic surfactant is TRITONX-405. The surfactant surprisingly reduces the non-specific backgroundfluorescence signal in the assay.

With the preferred anti-IgE solutions of this invention, the incubationtime of the solutions with the insoluble support is temperaturedependent. At temperatures of 18° to 40° C., incubation times of atleast from 30 to 180 minutes can be used. The preferred temperatures arewithin the range of from 20° to 26° C., and at these temperatures,incubation times from 60 to 120 minutes can be employed. It should beappreciated that prolonged incubation times in any of the steps of thisinvention can reduce the efficacy of the process. Since rapid analysisis an objective of this invention, the lowest times which still yieldthe desired accuracy are preferred.

The solid support is then rinsed to remove residual, unconjugated enzymelabeled anti-IgE antibody. The rinse solutions described above aresuitable.

The third step of the process of this invention comprises contacting thesolid support with a solution of a substrate which undergoes chemicalreaction in the presence of the fluorogenic enzyme for a time sufficientfor fluorescent compounds to be formed. Suitable substrates and theenzymes they are converted by are known in the art and are described inU.S. Pat. No. 4,190,496, for example. Examples of substrates have beendescribed hereinabove with respect to the corresponding fluorogenicenzyme.

The solid is contacted with an aqueous solution of the substratecontaining from 10⁻² to 10⁻¹⁰ molar and preferably from 10⁻⁴ to 10⁻⁵molar concentrations of the substrate. Preferred additional reagents andbuffers in the substrate solution include 2-amino-2-methyl-1-propanolbuffer and magnesium chloride, for example.

The substrate solution is incubated with the insoluble support forsufficient time for the fluorescent reaction product to form. Attemperatures of from 18° to 40° C., incubation times of from 5 to 240minutes can be used. Preferably, the temperature is within the range offrom 20° to 26° C., and the incubation time is from 30 to 90 minutes.

The fluorescence level in the solution is then measured. The equipmentand procedures for determining the level of fluorescence in thesubstrate solutions are those conventionally employed in the art. Thelevel of fluorescence is a function of the enzyme concentration on theinsoluble support which is, in turn, an inverse function of the amountof allergen in the diluted allergen extract. Suitable fluorometers arethe fluorometers by Perkin-Elmer, American Instrument Company, andTurner Designs. The Allergenetics Fluorometer (Allergenetics, Inc.,Mountain View, Calif.) is preferred.

In reverse inhibition assay procedures using serial dilution techniques,it is necessary to carry out steps of the method of this invention withtwo controls in addition to the carrying it out with diluted allergenextracts. For one control, the procedure is repeated without allergenextract to yield a fluorescence reading A, and for the other control,the procedure is again repeated without reagent allergen specific IgE toyield a fluorescence reading a. The percentage (%) of inhibition by eachdilution of allergen extract of unknown potency and by each dilution ofreference allergen extract of standard potency is then determined fromthe respective fluorescence readings X.

The percentage (%) of inhibition is calculated from the fluorescentreadings obtained according to the following equation: ##EQU1## whereinA is the fluorescence signal obtained in carrying out the proceduredescribed above without any allergen extract,

X is the fluorescence signal obtained with diluted allergen extract, and

a is a blank fluorescence signal obtained in carrying out the proceduredescribed above without any reagent allergen specific IgE.

Inhibition curves are established for the allergen extract of unknownpotency and for the reference allergen extract of known or standardpotency as shown in the Figure with the percentage (%) of inhibition onthe vertical axis and the amount of allergen extract (or dilutionfactor) on the horizontal axis (logarithymic scale) of logarithymicpaper.

To standardize a commercial allergen extract for use for desensitizingpatients experiencing allergic reactions, it is preferred to carry outthe above-described procedure with both the reference allergen extractand unknown allergen extract. An inhibition curve is established foreach extract with the (%) inhibition on the vertical axis and amount ofallergen extract or dilution factor on the horizontal axis. By comparingthe inhibition curves as illustrated in the Figure, the relativeconcentrations or dilutions of the allergen extract being standardizedand the standard extract necessary to provide the same level ofinhibition provides a highly accurate measure of relative (and absolute)potency. The range of concentrations is most advantageously obtained byserial dilution.

This invention is further illustrated by the following specific butnon-limiting examples. Temperatures are given in degrees Centigrade.

EXAMPLE 1

To a solution of timothy grass pollen allergen extract (3 mg/ml) wasadded 10 microliters of a 5 wt/% bovine serum albumin (BSA) solution.After addition, the solution was kept at 4° C., and 5 mg of1-Ethyl-3-(3-N,N-Dimethylaminopropyl)carbodiimide (ECDI) was added. Themixture was gently stirred at 4° C. for 20 minutes. The additions ofboth BSA and ECDI were repeated three more times. The final mixture wasallowed to stand at 4° C. overnight to yield a conjugate of timothygrass pollen allergen covalently bonded to BSA.

EXAMPLE 2

The procedure of Example 1 was repeated, replacing the timothy grasspollen extract with the following allergenic extracts: Grasses--BermudaGrass, Cynodon dactylon, Orchard Grass, Dactylis glomerata, PerennialRye Grass, Lolium perenne, June Grass (Kentucky Blue), Poa pratensis,Bent Grass, Agrostis maritima, Johnson Grass, Sorghum halepense, BromeGrass, Bromus inermis, Bahia Grass, Paspalum notatum, Corn Grass, Zeamays, Meadow Fescue, Festuca elatior, and Redtop, Agrostis alba;Weeds--Short Ragweed, Ambrosia artemisifolia, Western Ragweed; Ambrosiapsilostachya, False Ragweed, Franseria acanthicarpa; Sagebrush (common),Artemisia tridentata, Dandelion, Taraxacum vulgare, English Plantain,Plantago lanceolata, Lamb's Quarters, Chenopodium album, RussianThistle, Salsola kali, Goldenrod, Solidago sp., Pigweed, Amaranthusretroflexus, Dock (yellow), Rumex crispus, and Sheep Sorrel, Rumexacetosella; Trees--Box Elder (Maple), Acer negundo, Alder, Alnusrhombifolia, Birch, Betula nigra, Mountain Cedar, Juniperus sabinoides,White Oak, Quercus alba, Elm, Ulmus americana, Olive, Olea europaea,Black Walnut, Juglans nigra, Sycamore, Platanus occidentalis,Cottonwood, Populus trichocarpa, White Ash, Fraxinus americana, WhitePlain, Pinus monticola, Eucalyptus, Eucalyptus sp., Acacia, Acaciabaileyana, Aspen, Populus tremuloides, Arizona Cypress, Cupressusarizonica, Mesquite, Prosopis juliflora, Privet, Ligustrum ovalifolium,Melaleuca (Punk Tree), Melaleuca leucadendron, and Australian Pine(Beefwood), Casuarina equisetifolia; Epidermals--Cat Epithelium, DogHair and Dander, Horse Hair and Dander, Cow Hair and Dander, Guinea PigHair and Dander, Feather Mix (Chicken, Duck & Goose), and Wool (Sheep);Molds--Penicillium notatum, Cladosporium herbarum, Aspergillusfumigatus, Mucor racemosus, Candida albicans, and Alternaria tenuis;House Dust; Mite--Dermatophagoides farinae; and Foods--Milk, Wheat,Corn, Rice, Peanut, Soybean, Shrimp, Tomato, Pork, Carrot, Orange,Potato, Tuna, Beef, Lamb, Chicken, Whole Egg, Yeast (Bakers), SweetPotato, Cabbage, Lettuce, Pepper (Bell), Apple, Cranberry, Grape,Barley, and Onion. This yielded the corresponding, respective,covalently bonded BSA conjugate of each allergen.

EXAMPLE 3

Repeating the procedure of Example 1 but replacing the timothy grasspollen extracts with extracts of the following tree pollens yields thecorresponding, respective covalently bonded BSA-allergen conjugates:Acacia--Acacia longifolia; Ailanthus (See Tree of Heaven)--Ailanthusaltissima; Alder, Mountain (Tag) (Slender)--ainus tenuifolia/incana;Alder, Red (Oregon)--Alnus rubra; Alder, Sitka--Alnus sinuata;Almond--Prunus amygdalus; Apple--Pyrus malus (Malus pumila);Apricot--Prunus armeniaca; Arbor Vitae, Oriental (Ornamental)--Betulapapyrifera; Birch, Spring--Betula fontinalis; Birch, White(Weeping)--Betula pendula; Birch, Yellow--Betula lutea; Blue Beech (Am.Hornbeam)--Carpinus carolineana; Bottle Brush--Callistemon citrinus;Butternut--Juglans cinerea; Carob Tree--Ceratonia siliqua; Cedar,Deodar--Cedrus deodora; Cedar, Giant--Thuja plicata; Cedar,Incense--Linocedrus decurrens; Cedar, Japanese--Cryptomeria japonica;Cedar, Port Orford (Lawson Cypress)--Chamaecyparis lawsoniana; Cedar,Red--Juniperus virginiana; Cedar, Rocky Mountain--Juniperus scopulorum;Cedar, Salt (Tamarisk)--Tamarix gallica; Cedar, White--Thujaoccidentalis; Cherry, Prunus cerasus; Chestnut, American--Castaneadentata; Chestnut, Horse--Aesculus hippocastanum; Cottonwood,Common--Populus deltoides; Cottonwood, Fremont--Populus fremontii;Cypress, Bald (White)--Taxodium distichum; Cypress, Italian--Cupressussempervirens; Cypress, Monterey--Cupressus macrocarpa;Elderberry--Sambucus glauca; Elm, Cedar (Fall Blooming)--Ulmuscrassifolia; Elm, Chinese--Ulmus parvifolia; Elm, Siberian--Ulmuspumila; Elm, Slippery--Ulmus fulva (rubra); Fir, Douglas--Pseudotsugamenziesii; Fir, Red (Noble)--Abies nobilis (procera); Fir, White--Abiesconcolor; Gum, Sweet--Liquidambar styraciflua; Hackberry--Celtisoccidentalis; Hazelnut, American--Corylus americana; Hemlock,Eastern--Tsuga canadensis; Hemlock, Western--Tsuga heterophylla;Hickory, Shagbark--Carya ovata; Hickory, Shellbark-- Carya laciniosa;Hickory, White--Carya tomentosa; Ironwood (Hop-Hornbeam)--Ostryavirginiana; Juniper, California--Juniperus californica; Juniper,Chinese--Juniperus chinensis; Juniper, Oneseed--Juniperus monosperma;Juniper, Pinchot--Juniperus poinchotti; Juniper, Utah--Juniperusosteosperma (juniperus utahensis); Juniper, Western--Juniperusoccidentalis; Lilac--Syringa vulgaris; Linden (Basswood)--Tiliaamericana; Locust, Black--Robinia pseudoacacia; Maple, Big-Leaf(Coast)--Acer macrophyllum; Maple, Hard (Sugar)--Acer saccharum; Maple,Red--Acer rubrum; Maple, Soft (Silver)--Acer saccharinum; Mock Orange,Wild (Syringa)--Philadelphus lewisii; Mulberry, Paper--Broussonetiapapyifera; Mulberry, Red--Morus rubra; Mulberry, White--Morus alba; Oak,Arizona (Gambel)--Quercus gambelii; Oak, Arizona Scrub (Canyon)--Quercuschrysolepsis; Oak, Black (Yellow)--Quercus velutina; Oak, BlackJack--Quercus marilandica; Oak, Bur--Quercus macrocarpa; Oak, CaliforniaBlack--Quercus kelloggii--californica; Oak, California Scrub--Quercusdumosa; Oak, Coast Live--Quercus agrifolia; Oak, Engelmann--Quercusengelmanii; Oak, Garry (Western White)--Quercus garryana; Oak,Holly--Quercus ilex; Oak, Interior Live--Quercus wislizenii; Oak,Post--Quercus stellata; Oak, Red--Quercus rubra; Oak, Swamp(Pin)--Quercus palustris; Oak, Valley--Quercus lobata; Oak, VirginiaLive--Quercus virginiana; Oak, Water--Quercus nigra; Olive--Oleaeuropaea; Orange--Citrus sinensis; Osage Orange--Maclura pomifera; Palm,Date--Phoenix dactylifera; Palm, Dwarf--Chamaerops humulis; Palm, CanaryIsland Date (Ornamental)--Phoenix canariensis; Palm, Queen-- Cocosplumosa; Peach--Prunus persica; Pear--Pyrus communis; Pecan--Caryapecan; Pepper Tree, California--Schinus molle; Pepper Tree,Brazilian--Schinus terebinthifolius; Pine, Austrian--Pinus nigra; Pine,Canary Island--Pinus canariensis; Pine, Digger--Pinus sabiniana; Pine,Loblolly--Pinus taeda; Pine, Lodgepole--Pinus contorta; Pine,Monterey--Pinus radiata; Pine, Pinyon--Pinus edulis; Pine, Red(Norway)--Pinus resinosa; Pine Shortleaf--Pinus echinata; Pine, VirginiaScrub--Pinus, virginiana; Pine, Western Yellow (Ponderosa)--Pinusponderosa; Pine, White (Eastern)--Pinus strobus; Plum (Prune)--Prunusdomestica; Poplar, Balsam--Populus balsamifera; Poplar,Lombardy--Populus nigra-italica; Western Balsam (See Cottonwood, Black)Populus trichocarpa; Poplar, White--Populus alba; Privet--Ligustrumspp.; Redwood--Sequoia sempervirens; Russian Olive--Elaeagnusangustifolia; Spruce, Red--Picea rubens; Spruce, Sitka--Piceastitchensis; Sycamore, Mapleleaf--Platanus acerifolia; Sycamore,Western--Platanus racemosa; Tamarack (Larch)--Larix occidentalis;Tamarisk (See Cedar, Salt)--Tamarix gallica; Tree of Heaven--Ailanthusaltissima; Walnut, Arizona--Juglans rupestris; Walnut, Hind's CaliforniaBlack--Juglans hindsii; Walnut, So. California Black'Juglanscalifornica; Walnut, English--Juglans regia; Willow, Arroyo--Salixlasiolepis; Willow, Black--Salix nigra; Willow, Pussy--Salix discolor;Willow, Red--Salix laevigata; Willow, Yellow--Salix lasiandra.

EXAMPLE 4

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following grass and weed pollensyields the corresponding, respective covalently bonded BSA-allergenconjugates: Barley, Cultivated--Hordeum vulgare; Bluegrass, Annual--Poaannua; Bluegrass, Canada--Poa compressa; Bluegrass, Sandberg--Poasandbergii; Brome Broncho-Ripgut--Bromus rigidus; Brome,California--Bronus carinatus; Brome, Cheat--Bromus secalinus; Brome,Soft Cheat--Bromus mollis; Bunch, Blue (Northwestern Bunch)--Agropyronspicatum; Canarygrass--Phalaris canariensis; Canarygrass, Reed--Phalarisarundinacea; Fescue, Red--Festuca rubra; Grama Grass, Blue (SideOats)--Bouteloua gracilis; Koeler's Grass (Western Junegrass)--Koeleriacristata; Lovegrass, Hawaiian--Eragrostis variabilis; Oats, CommonCultivated--Avena sativa; Oatgrass, Tall--Avena elatior (Arrhenatherumelatius); Quack Grass--Agropyron repens; Rye, Cultivated--Secalecereale; Ryegrass, Alkali--Elymus triticoides; Ryegrass, GiantWild--Elymus cinereus; Ryegrass, Italian--Lolium multiflorum; Ryegrass,Western--Elymus glaucus; Salt Grass--Distichlis stricta; Sorghum, CommonCultivated--Sorghum vulgare; Sudan Grass--Sorghum vulgare var. sudanese;Sweet Vernal grass--Anthoxanthum odoratum; Velvetgrass--Holcus Ianatus;Wheat, Cultivated--Triticum aestivum; Wheatgrass, Crested--Agropyroncristatum; Wheatgrass, Western--Agropyron smithii; Alfalfa--Medicagosativa; Aster--Aster sinensis; Balsam Root--Balsamorhiza sagittata;Bassia--Bassia hyssopifolia; Beach Bur--Franseria bipinnatifida; BurroBrush (Greasebush)--Hymenoclea salsola; Careless Weed--Amaranthuspalmeri; Castor Bean--Ricinus communis; Cattail, Broadleaf--Typhalatifolia; Clover, Red--Trifolium pratense; Clover, Red--Trifoliumpratense; Clover, Sweet, Yellow--Melilotus officinalis; Clover, White(Dutch)--Trifolium repens (album); Cocklebur, Common--Xanthiumstrumarium; Cocklebur, Spiny--Xanthium spinosum; Cosmos--Cosmosbipinnatus; Daffodil--Narcissus pseudo-narcissus; Dahlia--Dahlia pinnatax coccinea; Daisy/Chrysanthemum (Oxeyed Daisy)--Chrysanthemumleucanthemum; Dock, Bitter--Rumex obtusifolius; Dog Fennel(Mayweed)--Anthemix cotula; Fireweed, Alaska--Epilobium angustifolium;Gladiolus--Gladiolus Xhortulanus; Greasewood--Sarcobatus vermiculatus;Hemp--Cannabis sativa; Hops--Humulus lupulus; Hopsage--Grayia spinosa;Iodine Bush (Burro Weed)--Allenrolfea occidentalis; Kochia (Mex.Firebush)--Kochia scoparia; Lily, Easter--Lilium longiflorum;Marigold--Tagetes patula; Marshelder, Burweed (Giant Poverty)--IvaXanthifolia; Marshelder, Narrowleaf (August)--Iva angustifolia;Marshelder, True (Rough)--Iva ciliata; Mexican Tea--Chenopodiumambrosioides; Mustard, Black--Brassica nigra; Mustard, CommonYellow--Brassica campestris; Nettle--Urtica dioica (gracilis);Pickleweed--Salicornia ambigua; Pigweed, Spiny--Amaranthus spinosus;Poppy, California--Eschoscholzia californica; Povertyweed, Small--Ivaaxillaris; Rabbit Brush--Chryso--thamnus nauseosus; Rabbit Bush (BurRagweed)--Franseria deltoides; Ragweed, Canyon--Franseria ambrosioides;Ragweed, Desert--Franseria dumosa; Ragweed, Giant--Ambrosia trifida;Ragweed, Silver--Dicoria canescens; Ragweed, Slender--Franseriatenuifolia; Ragweed, Southern--Ambrosia bidentata; Rose--Rosamultiflora; Sagebrush--Annual--Artemisia annua; Sagebrush,Coast--Artemisia californica; Sagebrush, Green (Tarragon)--Artemisiadracunculus; Sagebrush, Mugwort--Artemisia vulgaris heterophylla;Sagebrush, Pasture (Carpet)--Artemisi frigida; Sagebrush, SandDune--Artemisia pycnocephala; Sagebrush, White (Prairie)--ArtermisiaIudoviciana; Saltbush, Annual--Atriplex wrightii; Scale, All--Atriplexpolycarpa; Scale, Bract--Atriplex serenana bracteosa; Scale,Brewers--Atriplex lentiformis breweri; Scale, Lens--Atriplexlentiformis; Scale, Red--Atriplex rosea; Scale, Silver(Fogweed)--Atriplex argentea expansa; Scale, Spear--Atriplex patulahastata; Scale, Wing (Shad)--Atriplex canescen; Scotch Broom--Cytisusscoparius; Sea Blite, California--Suaeda californica; Sedge--Carexbarbara; Sheep Fat--Atriplex confertifolia; Snapdragon--Antirrhinummajus; Suaeda (See Sea Blite); Sugar Beet--Beta vulgaris;Sunflower--Helianthus annuus; Waterhemp, Western--Acnida tamariscina;Winter Fat--Eurotia lanata; Wormseed (Jerusalem Oak)--Chenopodium,botrys; Wormwood, Absinthe--Artemisia absinthium.

EXAMPLE 5

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following epidermals and glandularextracts yields the corresponding covalently bonded BSA-allergenconjugates: Camel Hair & Dander; Deer Hair & Dander; Feathers, Parakeet;Feathers, Pigeon; Feathers, Turkey; Fox Fur; Gerbil Hair & Epithelium;Glue, Fish; Goat Hair & Dander; Hamster Hair & Epithelium; Hog Hair &Dander; Human Hair; Mink Fur; Mohair; Monkey Hair & Epithelium; MouseHair & Epithelium; Poodle Hair & Dander; Pyrethrum; Rabbit Hair &Epithelium; Rat Hair & Epithelium; Seal Fur; Wool, Sheep.

EXAMPLE 6

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following dusts yields thecorresponding respective covalently bonded BSA-allergen conjugates:Acacia Gum; Alfalfa Hay; Algae, Chlorella spp.; Carragheen Gum; CoconutFiber; Cotton Linters; Cottonseed; Dust, Barley; Dust, Corn; Dust, GrainMill; Dust, Mattress; Dust, Oat; Dust, Pea; Dust, Rye; Dust, Soybean;Dust, Upholstery; Dust, Wheat; Dust, Wood--Cedar/Juniper; Dust,Wood--fir/Hemlock; Dust, Wood--Gum; Dust, Wood--Mahogany; Dust,Wood--Maple; Dust, Wood--Oak Mix; Dust, Wood--Pine Mix; Dust,Wood--Redwood; Dust, Wood--Spruce; Dust, Wood--Walnut; Fern Spores, sp.;Flax Fiber; Flaxseed; Hemp; Jute; Kapok; karaya Gum; Lycopodium; OrrisRoot; Paper Mix; Pyrethrum; Silk; Sisal; Tragacanth Gum; Timothy Hay;Tobacco, Pipe; Tobacco, Cigarette; Tobacco, Cigar; Tobacco, Leaf.

EXAMPLE 7

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following foods yields thecorresponding respective covalently bonded BSA-allergen conjugates:Allspice; Almond; Apricot, Food; Arrowroot; Artichoke; Asparagus;Avocado; Banana; Bay Leaf; Bean, Kidney; Bean, Lima; Bean, Navy; Bean,Pinto-Frijole; Bean, String/Wax; Beet; Black-Eyed Pea; Blueberry; BrazilNut; Buckwheat; Cashew Nut; Celery; Cheese, Cheddar (American); Cheese,Parmesan; Cheese, Roquefort; Cheese, Swiss; Cherry Mix; Chewing GumBase; chicken; Chicory; Chili Pepper; Chocolate/Cocoa; Cinnamon; Clam;Cloves; Cola; Coconut; Codfish Mix; Coffee; Crab; Cucumber; CurryPowder; Date; Dill; Egg White; Egg, Yolk; Eggplant; Endive; Garlic;Gelatine; Ginger; Raisin Mix; Grapefruit; Haddock; Halibut; Hazelnut(Filbert) Herring; Honey; Hops Food; Horseradish; Lamb; Lemon; Lentil;Lime; Liver; Beef (Calves); Lobster; Mackerel; Malt; Mangoes; Maple,Syrup/Sugar; Melon, (see Muskmelon Mix); Milk, Cow's (Albumin); Milk,Cow's (Casein); Milk, Cow's (Whey); Milk, (Evaporated); Milk, Goat's;Mint Mix (Peppermint/Spearmint); Mushroom; Mustard; Nutmeg; Oat, Whole(Grain); Okra; Olive Mix; Onion; Orange, Mandarin/Tangerine; Oregano;Oyster Mix; Papaya; Paprika; Parsley; Parsnip; Pea; Peach Food; PearFood; Pecan Food; Pepper, Black/-White; Pepper, Bell (Green/Red); Perch,Lake; Pineapple; Plum/Prune Mix; Poppy Seed; Pumpkin; Rabbit Meat;Radish; Raspberry; Snapper; Rhubarb; Rice, Wild; Rye, Whole (Grain);Safflower Seed; Sage; Salmon; Scallops; Sesame Seed; Sole; Spinach;Squash, Mix; Strawberry; Sugar (Beet); Sugar (Cane); Sunflower Seeds;Tapioca; Tea; Thyme; Trout; Turkey; Turnip; Vanilla; Walnut Food, Black;Walnut Food, English; Watermelon; Whitefish; Yeast, Brewers; Yeast Mix(Bakers/Brewers, Sacchoromyces cerevisiae).

EXAMPLE 8

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following molds yields thecorresponding respective covalently bonded BSA-allergen conjugates:Aspergillus clavatus; Aspergillus glaucus; Aspergillus nidulans;Aspergillus niger; Aspergillus restrictus; Aspergillus sydowi;Aspergillus terreus; Botrytis cinerea; Cephalosporium acremonium;Cephalothecium (Trichothecium) reseum; Chaetomium globosum; Cryptococcusterreus; Cunninghamella elegans; Curvularia spicifera; Dematium nigrum;Epicoccum nigrum; Epidermophyton floccosum; Fomes rimosus; Fusariumvasinfectum; Geotrichum candidum; Helminthosporium maydis;Helminthosporium; Hormodendrum (Cladosporium); Monilia sitophila;Mycogone sp.; Neurospora crassa; Nigrospora sphaerica; Oidiodendrum sp.;Paecilomyces varioti; Penicillium artramentosium; Penicillium biforme;Penicillium carminoviolaceum; Penicillium chrysogenum; Penicilliumdigitatum; Penicillium expansum; Penicillium glaucum; Penicilliumintricatum; Penicillium luteum; Penicillium roqueforti; Penicilliumroseum; Phoma herbarum; Pleospora sp.; Poria sp.; Pullularia pullulans;Rhizopus nigricans; Rhodotorula glutinis; Saccharomyces cerevisiae (SeeYeast Mix); Scopulariopsis brevicaulis; Spondylocladium sp.;Sporobolomyces salmonicolor; Stemphylium botryosum; Streptomycesgriseus; Trichoderma viride; Typhula idahoensis; Verticillum alboatrum.

EXAMPLE 9

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following smuts yields thecorresponding respective covalently bonded BSA-allergen conjugates:Smut, Barley; Smut, Bermuda; Smut Corn; Smut, Johnson; Smut, Oat; Smut,Sorghum; Smut, Wheat.

EXAMPLE 10

Repeating the procedure of Example 1 but replacing the timothy grasspollen extract with extracts of the following insects and insect venomsyields the corresponding covalently bonded BSA-allergen conjugates:Ants, (Black and Red); Ants, Carpenter; Ants, Fire; Aphid; Bee, Bumble;Bee, Honey; Blackfly; Butterfly; Caddis Fly; Cricket; Cockroach Mix;Deer Fly; Flea antigen; Fruit Flies; Gnat sp.; Horney, Black and Yellow;Horse Fly; House Fly; Mayfly sp.; Mite (D. farinae); Mosquito Mix; Moth,Miller; Wasp; Yellow Jacket; Honey Bee Venom Protein--Apis mellifera;Wasp Venom Protein--Polistes sp.; White-faced Horner VenomProtein--Dolichovespula maculata; Yellow Hornet VenomProtein--Dolichlovespula arenaria; Yellow Jacket Venom Protein--Vespulasp.; Mixed Vespid Venom Protein.

EXAMPLE 11

The timothy grass pollen allergens-conjugate with BSA prepared inExample 1 was diluted in phosphate buffer, pH 8.5. To each microtiterwell was added precisely 100 microliters of the diluted conjugatesolution. The coating process was allowed to proceed at room temperaturefor 2 hours (or overnight). At the end of the coating process, theliquid in each well was removed by aspiration. The wells were washedthree times (3×200 microliters) with a phosphate washing buffercontaining sorbitol and TRITON ×405. The wells thus coated can be usedfor assaying patient serum for timothy grass pollen allergen specificIgE antibodies.

EXAMPLE 12

The procedure of Example 11 was repeated but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example2. This yielded microtiter wells each having adhered thereto aBSA-allergen extract conjugate of: Grasses--Bermuda Grass, Cynodondactylon, Orchard Grass, Dactylis glomerata, Perennial Rye Grass, Loliumperenne, June Grass (Kentucky Blue), Poa pratensis, Bent Grass, Agrostismaritima, Johnson Grass, Sorghum halepense, Brome Grass, Bromos inermisBahia Grass, Paspalum notatum, Corn Grass, Zea mays, Meadow Fescue,Festuca elatior, and Redtop, Agrostis alba; Weeds--Short Ragweed,Ambrosia artemisifolia, Western Ragweed, Ambrosia psilostachya, FalseRagweed, Franseria acanthicarpa, Sagebrush (common), Artemisiatridentata, Dandelion, Taraxacum vulgare, English Plantain, Plantagolanceolata, Lamb's Quarters, Chenopodium album, Russian Thistle, Salsolakali, Goldenrod, Solidago sp., Pigweed, Amaranthus retroflexus, Dock(yellow), Rumex crispus, and Sheep Sorrel, Rumex acetosella; Trees--BoxElder (Maple), Acer negundo, Alder, Alnus rhombifolia, Birch, Betulanigra, Mountain Cedar, Juniperus sabinoides, White Oak, Quercus alba,Elm, Ulmus americana, Olive, Olea europaea, Black Walnut, Juglans nigra,Sycamore, Platanus occidentalis, Cottonwood, Populus trichocarpa, WhiteAsh, Fraxinus americana, White Pine, Pinus monticola, Eucalyptus,Eucalyptus sp., Acacia, Acacia baileyana, Aspen, Populus tremuloides,Arizona Cypress, Cupressus arizonica, Mesquite, Prosopis juliflora,Privet, Ligustrum ovalifolium, Melaleuca (Punk Tree), Melaleucaleucadendron, and Australian Pine (Beefwood), Casuarina equisetifolia;Epidermals--Cat Epithelium, Dog Hair and Dander; Horse Hair and Dander,Cow Hair and Dander, Guinea Pig Hair and Dander, Feather Mix (Chicken,Duck & Goose), and Wool (Sheep); Molds--Penicillium notatum,Cladosporium herbarum, Aspergillus fumigatus, Mucor racemosus, Candidaalbicans, and Alternaria tenuis; House Dust; Mite--Dermatophagoidesfarinae; and Foods--Milk, Wheat, Corn, Rice, Peanut, Soybean, Shrimp,Tomato, Pork, Carrot, Orange, Potato, Tuna, Beef, Lamb, Chicken, WholeEgg, Yeast (Bakers), Sweet Potato, Cabbage, Lettuce, Pepper (Bell),Apple, Cranberry, Grape, Barley, and Onion.

EXAMPLE 13

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example3 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of: Acacia--Acacia longifolia; Ailanthus (See Tree ofHeaven)--Ailanthus altissima; Alder, Mountain (Tag) (Slender)--ainustenuifolia/incana; Alder, Red (Oregon)--Alnus rubra; Alder, Sitka--Alnussinuata; Almond--Prunus amygdalus; Apple--Pyrus malus (Malus pumila);Apricot--Prunus armeniaca; Arbor Vitae, Oriental (Ornamental)--Betulapapyrifera; Birch, Spring--Betula fontinalis; Birch, White(Weeping)--Betula pendula; Birch, Yellow--Betula lutea; Blue Beech (Am.Hornbeam)--Carpinus carolineana; Bottle Brush--Callistemon citrinus;Butternut--Juglans cinerea; Carob Tree--Ceratonia siliqua; Cedar,Deodar--Cedrus deodora; Cedar, Giant--Thuja plicata; Cedar,Incense--Linocedrus decurrens; Cedar, Japanese--Cryptomeria japonica;Cedar, Port Orford (Lawson Cypress)--Chamaecyparis lawsoniana; Cedar,Red--Juniperus virginiana; Cedar, Rocky Mountain--Juniperus scopulorum;Cedar, Salt (Tamarisk)--Tamarix gallica; Cedar, White--Thujaoccidentalis; Cherry, Prunus cerasus; Chestnut, American--Castaneadentata; Chestnut, Horse--Aesculus hippocastanum; Cottonwood,Common--Populus deltoides; Cottonwood, Fremont--Populus fremontii;Cypress, Bald (White)--Taxodium distichum; Cypress, Italian--Cupressussempervirens; Cypress, Monterey--Cupressus macrocarpa;Elderberry--Sambucus glauca; Elm, Cedar (Fall Blooming)--Ulmuscrassifolia; Elm, Chinese--Ulmus parvifolia; Elm, Siberian--Ulmuspumila; Elm, Slippery--Ulmus fulva (rubra); Fir, Douglas--Pseudotsugamenziesii; Fir, Red (Noble)--Abies nobilis (procera); Fir, White--Abiesconcolor; Gum, Sweet--Liquidambar styraciflua; Hackberry--Celtisoccidentalis; Hazelnut, American--Corylus americana; Hemlock,Eastern--Tsuga canadensis; Hemlock, Western--Tsuga heterophylla;Hickory, Shagbark--Carya ovata; Hickory, Shellbark--Carya laciniosa;Hickory, White--Carya tomentosa; Ironwood (Hop-Hornbeam)--Ostryavirginiana; Juniper, California--Juniperus californica; Juniper,Chinese--Juniperus chinensis; Juniper, Oneseed--Juniperus monosperma;Juniper, Pinchot--Juniperus pinchotti; Juniper, Utah--Juniperusosteosperma (juniperus utahensis); Juniper, Western--Juniperusoccidentalis; Lilac--Syringa vulgaris; Linden (Basswood)--Tiliaamericana; Locust, Black--Robinia pseudoacacia; Maple, Big-Leaf(Coast)--Acer macrophyllum; Maple, Hard (Sugar)--Acer saccharum; Maple,Red--Acer rubrum; Maple, Soft (Silver)--Acer saccharinum; Mock Orange,Wild (Syringa)--Philadelphus lewisii; Mulberry, Paper--Broussonetiapapyifera; Mulberry, Red--Morus rubra; Mulberry, White--Morus alba; Oak,Arizona (Gambel)--Quercus gambelii; Oak, Arizona Scrub (Canyon)--Quercuschrysolepsis; Oak, Black (Yellow)--Quercus velutina; Oak, BlackJack--Quercus marilandica; Oak, Bur--Quercus macrocarpa; Oak, CaliforniaBlack--Quercus kelloggii-californica; Oak, California Scrub--Quercusdumosa; Oak, Coast Live--Quercus agrifolia; Oak, Engelmann--Quercusengelmanii; Oak, Garry (Western White)--Quercus garryana; Oak,Holly--Quercus ilex; Oak, Interior Live--Quercus wislizenii; Oak,Post--Quercus stellata; Oak, Red--Quercus rubra; Oak, Swamp(Pin)--Quercus palustris; Oak, Valley--Quercus lobata; Oak, VirginaLive--Quercus virginiana; Oak, Water--Quercus nigra; Olive--Oleaeuropaea; Orange--Citrus sinensis; Osage Orange--Maclura pomifera; Palm,Date--Phoenix dactylifera; Palm, Dwarf--Chamaerops humulis; Palm, CanaryIsland Date (Ornamental)--Phoenix canariensis; Palm, Queen--Cocosplumosa; Peach--Prunus persica; Pear--Pyrus communis; Pecan--Caryapecan; Pepper Tree, California--Schinus molle; Pepper Tree,Brazilian--Schinus terebinthifolius; Pine, Austrian--Pinus nigra; Pine,Canary Island--Pinus canariensis; Pine, Digger--Pinus sabiniana; Pine,Loblolly--Pinus taeda; Pine, Lodgepole--Pinus contorta; Pine,Monterey--Pinus radiata; Pine, Pinyon--Pinus edulis; Pine, Red(Norway)--Pinus resinosa; Pine, Shortleaf--Pinus echinata; Pine,Virginia Scrub--Pinus virginiana; Pine, Western Yellow(Ponderosa)--Pinus ponderosa; Pine, White (Eastern)--Pinus strobus; Plum(Prune)--Prunus domestica; Poplar, Balsam--Populus balsamifera; Poplar,Lombardy--Populus nigra-italica; Western Balsam (See Cottonwood, Black)Populus trichocarpa; Poplar, White--Populus alba; Privet--Ligustrumspp.; Redwood--Sequoia sempervirens; Russian Olive--Elaeagnusangustifolia; Spruce, Red--Picea rubens; Spruce, Sitka--Piceasitchensis; Sycamore, Mapleleaf--Platanus acerifolia; Sycamore,Western--Platanus racemosa; Tamarack (Larch)--Larix occidentalis;Tamarisk (See Cedar, Salt)--Tamarix gallica; Tree of Heaven--Ailanthusaltissima; Walnut, Arizona--Juglans rupestris; Walnut, Hind's CaliforniaBlack--Juglans hindsii; Walnut, So. California Black--Juglanscalifornica; Walnut, English--Juglans regia; Willow, Arroyo--Salixlasiolepis; Willow, Black--Salix nigra; Willow, Pussy--Salix discolor;Willow, Red--Salix laevigata; Willow, Yellow--Salix lasiandra.

EXAMPLE 14

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example4 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Barley, Cultivated--Hordeum vulgare; Bluegrass,Annual--Poa annua; Bluegrass, Canada--Poa compressa; Bluegrass,Sandberg--Poa sandbergii; Brome Broncho-Ripgut--Bromos rigidus; Brome,California--Bromus carinatus; Brome, Cheat--Bromos secalinus; Brome,Soft Cheat--Bromus mollis; Bunch, Blue (Northwestern Bunch)--Agropyronspicatum; Canarygrass--Phalaris canariensis; Canarygrass, Reed--Phalarisarundinacea; Fescue, Red--Festuca rubra; Grama Grass, Blue (SideOats)--Bouteloua gracilis; Koeler's Grass (Western Junegrass)--Koeleriacristata; Lovegrass, Hawaiian--Eragrostis variabilis; Oats, CommonCultivated--Avena sativa; Oatgrass, Tall--Avena elatior (Arrhenatherumelatius); Quack Grass--Agropyron repens; Rye, Cultivated--Secalecereale; Ryegrass, Alkali--Elymus triticoides; Ryegrass, GiantWild--Elymus cinereus; Ryegrass, Italian--Lolium multiflorum; Ryegrass,Western--Elymus glaucus; Salt Grass--Distichlis stricta; Sorghum, CommonCultivated--Sorghum vulgare; Sudan Grass--Sorghum vulgare var. sudanese;Sweet Vernal grass--Anthoxanthum odoratum; Velvetgrass--Holcus Ianatus;Wheat, Cultivated--Triticum aestivum; Wheatgrass, Crested--Agropyroncristatum; Wheatgrass, Western--Agropyron smithii; Alfalfa--Medicagosativa; Aster--Aster sinensis; Balsam Root--Balsamorhiza sagittata;Bassia--Bassia hyssopifolia; Beach Bur--Franseria bipinnatifida; BurroBrush (Greasebush)--Hymenoclea salsola; Careless Weed--Amaranthuspalmeri; Castor Bean--Ricinus communis; Cattail, Broadleaf--Typhalatifolia; Clover, Red--Trifolium pratense; Clover, Sweet,Yellow--Melilotus officinalis; Clover, White (Dutch)--Trifolium repens(album); Cocklebur, Common--Xanthium strumarium; Cocklebur,Spiny--Xanthium spinosum; Cosmos--Cosmos bipinnatus; Daffodil--Narcissuspseudo-narcissus; Dahlia--Dahlia pinnata x coccinea; Daisy/Chrysanthemum(Oxeyed Daisy)--Chrysanthemum leucanthemum; Dock, Bitter--Rumexobtusifolius; Dog Fennel (Mayweed)--Anthemix cotula; Fireweed,Alaska--Epilobium angustifolium; Gladiolus--Gladiolus Xhortulanus;Greasewood--Sarcobatus vermiculatus; Hemp--Cannabis sativa;Hops--Humulus lupulus; Hopsage--Grayia spinosa; Iodine Bush (BurroWeed)--Allenrolfea occidentalis; Kochia (Mex. Firebush)--Kochiascoparia; Lily, Easter--Lilium longiflorum; Marigold--Tagetes patula;Marshelder, Burweed (Giant Poverty)--Iva Xanthifolia; Marshelder,Narrowleaf (August)--Iva angustifolia; Marshelder, True (Rough)--Ivaciliata; Mexican Tea--Chenopodium ambrosioides; Mustard, Black--Brassicanigra; Mustard, Common Yellow--Brassica campestris; Nettle--Urticadioica (gracilis); Pickleweed--Salicornia ambigua; Pigweed,Spiny--Amaranthus spinosus; Poppy, California--Eschoscholziacalifornica; Povertyweed, Small--Iva axillaris; RabbitBrush--Chryso-thamnus nauseosus; Rabbit Bush (Bur Ragweed)--Franseriadeltoides; Ragweed, Canyon--Franseria ambrosioides; Ragweed,Desert--Franseria dumosa; Ragweed, Giant--Ambrosia trifida; Ragweed,Silver--Dicoria canescens; Ragweed, Slender--Franseria tenuifolia;Ragweed, Southern--Ambrosia bidentata; Rose--Rosa multiflora;Sagebrush-Annual--Artemisia annua; Sagebrush, Coast--Artemisiacalifornica; Sagebrush, Green (Tarragon)--Artemisia dracunculus;Sagebrush, Mugwort--Artemisia vulgaris heterophylla; Sagebrush, Pasture(Carpet)--Artemisi frigida; Sagebrush, Sand Dune--Artemisiapycnocephala; Sagebrush, White (Prairie)--Artemisia Iudoviciana;Saltbush, Annual--Atriplex wrightii; Scale, All--Atriplex polycarpa;Scale, Bract--Atriplex serenana bracteosa; Scale, Brewers--Atriplexlentiformis breweri; Scale, Lens--Atriplex lentiformis; Scale,Red--Atriplex rosea; Scale, Silver (Fogweed)--Atriplex argentea expansa;Scale, Spear--Atriplex patula hastata; Scale, Wing (Shad)--Atriplexcanescen; Scotch Broom--Cytisus scoparius; Sea Blite, California--Suaedacalifornica; Sedge--Carex barbara; Sheep Fat--Atriplex confertifolia;Snapdragon--Antirrhinum majus; Suaeda (See Sea Blite); Sugar Beet--Betavulgaris; Sunflower--Helianthus annuus; Waterhemp, Western--Acnidatamariscina; Winter Fat--Eurotia lanata; Wormseed (JerusalemOak)--Chenopodium botrys; Wormwood, Absinthe--Artemisia absinthium.

EXAMPLE 15

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example5 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Camel Hair & Dander; Deer Hair & Dander; Feathers,Parakeet; Feathers, Pigeon; Feathers, Turkey; Fox Fur; Gerbil Hair &Epithelium; Glue, Fish; Goat Hair & Dander; Hamster Hair & Epithelium;Hog Hair & Dander; Human Hair; Mink Fur; Mohair; Monkey Hair &Epithelium; Mouse Hair & Epithelium; Poodle Hair & Dander; Pyrethrum;Rabbit Hair & Epithelium; Rat Hair & Epithelium; Seal Fur; Wool, Sheep.

EXAMPLE 16

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example6 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Acacia Gum; Alfalfa Hay; Algae, Chlorella spp.;Carragheen Gum; Coconut Fiber, Cotton Linters; Cottonseed; Dust, Barley;Dust, Corn; Dust, Grain Mill; Dust, Mattress; Dust, Oat; Dust, Pea;Dust, Rye; Dust, Soybean; Dust, Upholstery; Dust, Wheat; Dust,Wood--Cedar/Juniper; Dust, Wood--fir/Hemlock; Dust, Wood--Gum; Dust,Wood--Mahogany; Dust, Wood--Maple; Dust, Wood--Oak Mix; Dust, Wood--PineMix; Dust, Wood--Redwood; Dust, Wood--Spruce; Dust, Wood--Walnut; FernSpores; sp.; Flax Fiber; Flaxseed; Hemp; Jute; Kapok; karaya Gum;Lycopodium; Orris Root; Paper Mix; Pyrethrum; Silk; Sisal; TragacanthGum; Timothy Hay; Tobacco, Pipe; Tobacco, Cigarette; Tobacco, Cigar; orTobacco, Leaf.

EXAMPLE 17

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example7 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Allspice; Almond; Apricot Food; Arrowroot;Artichoke; Asparagus; Avocado; Banana; Bay Leaf; Bean, Kidney; Bean,Lima; Bean, Navy; Bean, Pinto-Frijole; Bean, String/Wax; Beet;Black-Eyed Pea; Blueberry; Brazil Nut; Buckwheat; Cashew Nut; Celery;Cheese, Cheddar (American); Cheese, Parmesan; Cheese, Roquefort; Cheese,Swiss; Cherry Mix; Chewing Gum Base; chicken; Chicory; Chili Pepper;Chocolate/Cocoa; Cinnamon; Clam; Cloves; Cola; Coconut; Codfish Mix;Coffee; Crab; Cucumber; Curry Powder; Date; Dill; Egg White; Egg, Yolk;Eggplant; Endive; Garlic; Gelatine; Ginger; Raisin Mix; Grapefruit;Haddock; Halibut; Hazelnut (Filbert); Herring; Honey; Hops Food;Horseradish; Lamb; Lemon; Lentil; Lime; Liver, Beef (Calves); Lobster;Mackerel; Malt; Mangoes; Maple, Syrup/Sugar; Melon, (see Muskmelon Mix);Milk, Cow's (Albumin); Milk, Cow's (Casein); Milk, Cow's (Whey); Milk,(Evaporated); Milk, Goat's; Mint Mix (Peppermint/Spearmint); Mushroom;Mustard; Nutmeg; Oat, Whole (Grain); Okra; Olive Mix; Onion; Orange,Mandarin/Tangerine; Oregano; Oyster Mix; Papaya; Paprika; Parsley;Parsnip; Pea; Peach Food; Pear Food; Pecan Food; Pepper, Black/-White;Pepper, Bell (Green/Red); Perch, Lake; Pineapple; Plum/Prune Mix; PoppySeed; Pumpkin; Rabbit Meat; Radish; Raspberry; Snapper; Rhubarb; Rice,Wild; Rye, Whole (Grain); Safflower Seed; Sage; Salmon; Scallops; SesameSeed; Sole; Spinach; Squash, Mix; Strawberry; Sugar (Beet); Sugar(Cane); Sunflower Seeds; Tapioca; Tea; Thyme; Trout; Turkey; Turnip;Vanilla; Walnut Food, Black; Walnut Food, English; Watermelon;Whitefish; Yeast, Brewers; Yeast Mix (Bakers/Brewers, Sacchoromycescerevisiae).

EXAMPLE 18

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example8 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Aspergillus clavatus; Aspergillus glaucus;Aspergillus nidulans; Aspergillus niger; Aspergillus restrictus;Aspergillus sydowi; Aspergillus terreus; Botrytis cinerea;Cephalosporium acremonium; Cephalothecium (Trichothecium) reseum;Chaetomium globosum; Cryptococcus terreus; Cunninghamella elegans;Curvularia spicifera; Dematium nigrum; Epicoccum nigrum; Epidermophytonfloccosum; Fomes rimosus; Fusarium vasinfectum; Geotrichum candidum;Helminthosporium maydis; Helminthosporium; Hormodendrum (Cladosporium);Monilia sitophila; Mycogone sp.; Neurospora crassa; Nigrosporasphaerica; Oidiodendrum sp.; Paecilomyces varioti; Pencilliumartramentosum; Penicillium biforme; Penicillium carminoviolaceum;Penicillium chrysogenum; Penicillium digitatum; Penicillium expansum;Penicillium glaucum; Penicillium intricatum; Penicillium lutuem;Penicillium rogueforti; Penicillium roseum; Phoma herbarum; Pleosporasp.; Poria sp.; Pullularia pullulans; Rhizopus nigricans; Rhodotorulaglutinus; Saccharomyces cerevisiae (See Yeast Mix); Scopulariopsisbrevicaulis; Spondylocladium sp.; Sporobolomyces salmonicolor;Stemphylium botryosum; Streptomyces griseus; Trichoderma viride; Typhulaidahoensis; Verticillum alboatrum.

EXAMPLE 19

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example9 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Smut, Barley; Smut, Bermuda; Smut Corn; Smut,Johnson; Smut, Oat; Smut, Sorghum; or Smut, Wheat.

EXAMPLE 20

Repeating the procedure of Example 11 but replacing the timothy grasspollen extract--BSA conjugate with the BSA conjugate products of Example10 yields microtiter wells each having adhered thereto a BSA-allergenextract conjugate of Ants, (Black and Red); Ants, Carpenter; Ants, Fire;Aphid; Bee, Bumble; Bee, Honey; Blackfly; Butterfly; Caddis Fly;Cricket; Cockroach Mix; Deer Fly; Flea antigen; Fruit Flies; Gnat sp.;Horney, Black and Yellow; Horse Fly; House Fly; Mayfly sp.; Mite (D.farinae); Mosquito Mix; Moth, Miller; Wasp; Yellow Jacket; Honey BeeVenom Protein--Apis mellifera; Wasp Venom Protein--Polistes sp.;White-faced Horner Venom Protein--Dolichovespula maculata; Yellow HornetVenom Protein--Dolichovespula arenaria; Yellow Jacket VenomProtein--Vespula sp.; or Mixed Vespid Venom Protein.

EXAMPLE 21

In this procedure, timothy grass pollen allergen extract of unknownpotency (Unknown) is standardized against timothy grass pollen allergenextract reference standard (Standard) obtained from the United StatesBureau of Biologies.

Three-fold serial dilutions of the Unknown and Standard are prepared bystandard procedures from extracts which have been reconstituted from thedry (lyophilized) state with 5 ml distilled water per vial. A diluenthaving the following composition is used for the serial dilution:

    ______________________________________                                        PO.sub.4 buffer solution (PBS), pH 7.5                                                                  500    ml                                           BSA                       1      g                                            Non-ionic surfactant (Tween 20), 1% v/v                                                                 5      ml                                           Sodium azide, 10 wt. % solution                                                                         0.5    ml                                           ______________________________________                                    

The serial dilutions were prepared and mixed with serum containingtimothy allergen specific IgE (obtained from an atopic patientevidencing timothy pollen allergy) to make dilution sets in microtiterwells for the Standard corresponding to the following TABLE:

                                      TABLE.sup.(a)                               __________________________________________________________________________    Tube No.  1  2  3  4  5  6   7   8   9   10   11   12                         __________________________________________________________________________    Dilution factor                                                                          0 1/3                                                                              1/9                                                                              1/27                                                                             1/81                                                                             1/243                                                                             1/729                                                                             1/2187                                                                            1/6561                                                                            1/19683                                                                            1/59049                                                                             0                         Extract    0 200                                                                              200                                                                              200                                                                              200                                                                              200 200 200 200 200  200   0                         Diluent   400                                                                              400                                                                              400                                                                              400                                                                              400                                                                              400 400 400 400 400  400  400                        Total volume                                                                            400                                                                              600                                                                              600                                                                              600                                                                              600                                                                              600 600 600 600 600  600  400                        Volume added to                                                                          0 200                                                                              200                                                                              200                                                                              200                                                                              200 200 200 200 200    200.sup.(b)                                                                       0                         next tube                                                                     Volume of diluted                                                                       400                                                                              400                                                                              400                                                                              400                                                                              400                                                                              400 400 400 400 400  400  400                        extract                                                                       Serum added                                                                              80                                                                               80                                                                               80                                                                               80                                                                               80                                                                               80  80  80  80  80   80    80.sup.(c)               Buffer diluent added                                                                    320                                                                              320                                                                              320                                                                              320                                                                              320                                                                              320 320 320 320 320  320  320                        Total final volume                                                                      800                                                                              800                                                                              800                                                                              800                                                                              800                                                                              800 800 800 800 800  800  800                        __________________________________________________________________________     .sup.(a) All volumes are in microliters                                       .sup.(b) 200 microliters are discarded                                        .sup.(c) HSA added to provide serum without timothy pollen allergen           specific IgE.                                                            

The procedure is repeated with tube Nos. 13 to 24 with the Unknown toobtain a corresponding serial dilution act.

A microtiter well product prepared is described in Example 11 havingwells to which a BSA conjugate of timothy grass pollen allergen isadhered is soaked for 3-5 minutes with a buffered rinse solutioncontaining 0.85 wt. % sodium chloride, 0.02 wt. % TRITON ×405, 0.01 wt.% BSA, and 0.1 wt. % sodium azide preservative in a 0.0002M aqueousphosphate buffer solution, pH 7.2, and the surplus is removed.

The buffered rinse solution is prepared by diluting the followingconcentrate with 50 parts by volume distilled or deionized water to onepart by volume concentrate:

    ______________________________________                                        Bovine serum albumin    0.5    wt. %                                          Non-ionic surfactant (TRITON X-405)                                                                   0.1    wt. %                                          Sodium Chloride         17     wt. %                                          Sodium azide            2      wt. %                                          Sodium phosphate        0.01   M                                              pH adjusted to          7.4                                                   ______________________________________                                    

Serum IgE specific antibody for timothy grass pollen allergen isconjugated to the microtiter plate well surfaces.

To microtiter plate wells 1 to 12 to which a BSA conjugate of timothygrass pollen allergen is adhered is added with mixing 100 microliters ofdiluted reference Standard extract from a correspondingly numberedserial dilution tube. The procedure is repeated with microtiter platewells 13 to 24 using serially diluted Unknown extracts from thecorrespondingly numbered tubes. The mixtures are incubated for 2 hr atroom temperature, aspirated and washed three times with buffered rinsesolution.

The microtiter plate wells are then contacted with 100 microliters of asolution of alkaline phosphatase conjugated anti-human IgE monoclonalantibody prepared according to a modified procedure of M. O'Sullivan, etal, Analytical Biochem., vol. 100, page 100(1979). The monoclonalantibody is applied in a solution of 0.01M phosphate buffered saline, pH7.2, containing 4 wt.% polyethylene glycol having a molecular weight of4000 (PEG 4000), 0.05 wt.% TRITON X-405, 0.1 wt. % BSA, and 0.1 wt.%sodium azide preservative. The alkaline phosphatase conjugatedanti-human IgE monoclonal antibody solution is removed from themicrotiter plate wells, and they are rinsed three times with thebuffered rinse solution described above.

To each microtiter plate well is then added 100 microliters of asubstrate solution containing 10⁻⁴ M 4-methyl umbelliferyl phosphate in1.25M 2-amino-2-methylpropanol, pH 9.5 in deionized water containing0.125 mM magnesium chloride and 0.1 wt.% sodium azide. After 60 minutes,the fluorescence level in each well is read with a fluorometer with theexcitation at 365 nm and the reading at 450 nm.

The percentage (%) of inhibition of each dilution of Unknown andStandard is determined using the equations ##EQU2## wherein A is thefluorescence signal obtained in carrying out the procedure describedabove without any allergen extract,

X is the fluorescence signal obtained with diluted allergen extract, and

a is a blank fluorescence signal obtained in carrying out the proceduredescribed above without any reagent allergen specific IgE.

The (%) inhibitions are as follows:

    ______________________________________                                        Percentage (%) of Inhibition                                                  Timothy Pollen Allergens                                                      Dilution       Unknown   Standard                                             ______________________________________                                        1/3            94.8                                                           1/9            95.4      99                                                   1/27           93.5      97.4                                                 1/81           88.0      93.4                                                 1/243          80.5      87.9                                                 1/729          76.0      81.3                                                 1/2187         53.4      60.5                                                 1/6561         42.6      44.5                                                 1/19683        24.0      30.8                                                 1/59049        10.0      12.5                                                 ______________________________________                                    

The plot of the (%) inhibitions is shown in the Figure.

By comparing log plots in the Figure of the fluorescence levelsmeasured, as a function of extract concentration (dilution) of theStandard and Unknown extracts, the comparative potency is determined.

EXAMPLE 22

The procedure of Example 21 is repeated with the microtiter plate wellproducts of Examples 12 through 20, inclusive, the respective Standardand Unknown extracts, and with allergen specific reagent IgE specificthereto, the relative potencies of the respective Unknowns aredetermined.

The invention claimed is:
 1. A method for measuring the potency ofallergen extracts comprising the sequential steps of(a) contacting aninsoluble support having reagent allergen adhered thereto with anaqueous solution containing a predetermined amount of allergen extract,and an amount of IgE specific to allergenic components present in thereagent allergen and presumed present in the allergen extract which isinsufficient to bind with all of the allergenic components present, fora sufficient time to permit conjugation of IgE with allergeniccomponents, and removing the solution from the support; (b) contactingthe insoluble support with an aqueous solution of anti-IgE antibodylabeled with a fluorogenic enzyme for sufficient time to permitconjugation of anti-IgE antibody with IgE which has conjugated withreagent allergen on the insoluble support, and removing the solutionfrom the support; (c) contacting the insoluble support with an aqueoussolution of a substrate which undergoes chemical reaction to yield afluorescent product when in the presence of the fluorogenic enzyme, forsufficient time to yield a fluorescent product; and (d) measuring thelevel of fluorescence in the solution.
 2. The method of claim 1 whereinthe insoluble support has a plurality of reaction wells separated byopaque material.
 3. The method of claim 2 wherein the insoluble supportis a microtiter plate made of opaque material.
 4. The method of claim 1wherein the anti-IgE antibody is a monoclonal antibody.
 5. The method ofclaim 1 wherein the anti-IgE antibody is labeled with alkalinephosphatase.
 6. The method of claim 5 wherein the substrate is4-methyl-umbelliferyl phosphate.
 7. The method of claim 3 wherein themicrotiter plate is polystyrene or a styrene-(vinyl monomer) copolymer.8. The method of claim 1 wherein the insoluble support is contacted instep (b) with anti-IgE antibody in an aqueous solution containing from 1to 8 wt.% polyethylene glycol having a molecular weight within the rangeof from 1000 to 10,000.
 9. The method of claim 8 wherein the aqueoussolution contains from 0.001 to 0.5 wt. % of a non-ionic surfactant. 10.The method of claim 9 wherein the non-ionic surfactant isoctylphenoxypolyethoxyethanol.
 11. The method of claim 1 wherein theinsoluble support is contacted in step (b) with anti-IgE antibody in anaqueous solution containing from 0.001 to 0.5 wt. % of a non-ionicsurfactant.
 12. The method of claim 1 wherein the solution is removedfrom the insoluble support in step (a) by rinsing with a phosphatebuffered solution having a pH within the range of from 6 to 8 andcontaining a non-ionic surfactant.
 13. The method of claim 1 wherein theunconjugated anti-IgE antibody is removed from the insoluble support instep (b) with a phosphate buffered solution having a pH within the rangeof from 6 to 8 and containing a non-ionic surfactant.
 14. The method ofclaim 1 wherein the allergen adhering to the insoluble support is acovalently bonded conjugate of the allergen and a soluble protein oramino acid polymer.
 15. The method of claim 14 wherein the allergenadhering to the insoluble support is a covalently bonded conjugate ofthe allergen and a soluble animal protein.
 16. The method of claim 15wherein the insoluble support is prerinsed with an aqueous bufferedsolution containing from 0.005 to 2.5 wt. % of the animal protein beforebeing contacted with patient serum.
 17. The method of claim 15 whereinthe soluble animal protein is bovine serum albumin.
 18. The method ofclaim 17 wherein the insoluble support is prerinsed with an aqueousbuffered solution containing from 0.005 to 2.5 wt. % of bovine serumalbumin before being contacted with patient serum.
 19. The method ofclaim 15 wherein the allergen is covalently bonded to the bovine serumalbumin with a carbodiimide.
 20. The method of claim 15 wherein theallergen is covalently bonded to the bovine serum albumin with abifunctional crosslinking agent.
 21. The method of claim 1 wherein theallergen is derived from a pollen, mold, smut, animal dander, animalepidermal, insect, insect venom, dust, or food.
 22. The method of claim1 comprising(a) contacting an opaque polystyrene or styrene-(vinylmonomer) copolymer support having a reagent allergen-soluble proteinconjugate adhering thereto with an aqueous solution containing apredetermined amount of allergen extract and an amount of IgE specificto allergenic components present in the reagent allergen and presumedpresent in the allergen extract which is insufficient to bind with allof the allergenic components present, for a sufficient time to permitconjugation of IgE with allergenic components; (b) removing residualsolution from the support; (c) contacting the support with an anti-IgEantibody labeled with a fluorogenic enzyme in an aqueous solutioncontaining polyethylene glycol and a non-ionic surfactant for asufficient time to permit conjugation of anti-IgE antibody to anyallergen specific IgE conjugated to the support; (d) removing residualsolution from the support; (e) contacting the support with a solution ofa substrate which undergoes reaction in the presence of the fluorogenicenzyme to yield fluorescent product; and (f) measuring the fluorescencelevel of the solution.
 23. The method of claim 1 wherein steps (a)through (d) are repeated with a corresponding allergen of known potencyand the percentage of inhibitions corresponding to the fluorescencelevels are compared to determine the relative potency of the allergenextract being measured.